Multivalent Vaccines Comprising Recombinant Viral Vectors

ABSTRACT

The invention relates to vaccines comprising recombinant vectors, such as recombinant adenoviruses. The vectors comprise heterologous nucleic acids encoding for at least two antigens from one or more tuberculosis-causing bacilli. The invention also relates to the use of specific protease recognition sites linking antigens through which the encoded antigens are separated upon cleavage. After cleavage, the antigens contribute to the immune response in a separate manner. The recombinant vectors may comprise a nucleic acid encoding the protease cleaving the linkers and separating the antigens. The invention furthermore relates to the use of genetic adjuvants encoded by the recombinant vectors, wherein such genetic adjuvants may also be cleaved through the presence of the cleavable linkers and the specific protease.

FIELD OF THE INVENTION

The invention relates to the field of recombinant DNA and viral vectorvaccines. Specifically, it relates to recombinant DNA and viral vectorsharbouring nucleic acids encoding multiple antigens and/or adjuvants.

BACKGROUND OF THE INVENTION

Tuberculosis (TB) has been a major worldwide threat to human health forseveral thousands of years. TB caused by Mycobacterium tuberculosis isan infectious disease of the lung caused by infection through exposureto air-borne M. tuberculosis bacilli. These bacilli are extremelyinfectious and it has been estimated that currently approximatelyone-third of the world population (2 billion people) are infected. Ithas been further estimated that TB kills over 2 million people worldwideon an annual basis. Only 5 to 10% of the immunocompetent humans aresusceptible to TB, and over 85% of them will develop the diseaseexclusively in the lungs, while HIV-infected humans may also developsystemic diseases that will more easily lead to death.

Approximately 90% of M. tuberculosis-infected humans will not developthe disease. However, in these latently infected individuals the bacillican survive for many years and become reactivated for instance in thecase of a weakened immune system, such as after an HIV infection. Due tothe latent nature, infected individuals generally have to be treated byadministration of several antibiotics for up to 12 months, which is nota very attractive treatment in general and due to costs and the possibleoccurrence of multi-drug resistance, while it is also not a veryeffective treatment in most developing countries.

One relatively successful TB vaccine has been developed: the bacilliCalmette-Guerin (BCG) vaccine was generated in the early years of thetwentieth century, and was first given to individuals in 1921. The BCGvaccine is an attenuated strain of bacteria based on a Mycobacteriumbovis isolate obtained from a cow. It is a relatively safe vaccine,which is easy and rather cheaply produced. In the year 2000, BCGvaccination covered 86% of the world population. However, the vaccineappears to be not extremely effective for adult pulmonary TB and manyregions in developing countries still have very high rates of TB,despite the BCG-vaccine programs. It has been estimated that BCG vaccineprevents only 5% of all vaccine-preventable deaths by TB (Kaufmann,2000).

Due to the rather low protection rate of the BCG vaccine in general anddue to the specific protection in respect to childhood and disseminatedTB, more efforts were put in the development of new, more broadlyapplicable, vaccines against TB, based on other systems and knowledgeacquired in other fields such as vaccination against other tropicalinfectious diseases and HIV (review by Wang and Xing, 2002).

Different approaches were taken to develop new TB vaccines, ranging fromsubunit vaccines and DNA vaccines to modified mycobacterium strains.Moreover, also recombinant viral-based vaccines were generated, enablingthe transfer of M. tuberculosis antigens to antigen-presenting cellsthrough gene delivery vehicles such as Modified Vaccinia Ankara (MVA)vectors and replication-defective adenovirus vectors.

Naked DNA vaccines against TB have been described in WO 96/15241 (seealso EP 0792358), whereas many reports describe the use of numerousantigens from Mycobacterium tuberculosis in either recombinant orpurified form for their application in vaccines: WO 95/01441, WO95/14713, WO 96/37219, U.S. Pat. No. 6,599,510, WO 98/31388, WO98/44119, WO 99/04005, WO 99/24577, WO 00/21983, WO 01/04151, WO01/79274, WO 2004/006952, US 2002/0150592. The use of fusion proteinscomprising different TB antigens has also been suggested: See WO98/44119, EP 0972045 and EP 1449922, disclosing the use of a fusionpolypeptide between ESAT-6 and MPT59 (MPT59 is also referred to as Ag85Bor the 85B antigen).

Despite all these and other efforts in generating a vaccine againsttuberculosis that ensures both a strong cellular, a strong humoralresponse as well as a long-lasting high protection rate, no such vaccineis yet available.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Map of pAdApt35Bsu.myc

FIG. 2: Map of pAdApt35Bsu.TB.LM

FIG. 3: Map of pAdApt35Bsu.TB.SM

FIG. 4: Map of pAdApt35Bsu.TB.FLM

FIG. 5: Map of pAdApt35Bsu.TB.3M

FIG. 6: Map of pAdApt35Bsu.TB.4M

FIG. 7: Map of pAdApt35Bsu.TB.5M

FIG. 8: Map of pAdApt35Bsu.TB.6M

FIG. 9: Map of pAdApt35Bsu.TB.7M

FIG. 10. Western blot with anti-TB antigen polyclonal on lysates fromA549 cells infected with Ad35 viruses comprising nucleic acids encodingdifferent sets of TB antigens with the myc-tag (A) and without themyc-tag (B). (C) similar to (B), with molecular weight markers. See fornotation Table I.

FIG. 11. Experimental design of immunization protocol using 7 differentadenoviral vectors (DNA) harbouring different sets of nucleic acidsencoding tuberculosis antigens.

FIG. 12. Percentages of antigen-specific splenocytes that stain positivefor interferon-gamma production (IFNγ+) upon stimulation with no peptide(A: CD4+ cells, B: CD8+ cells).

FIG. 13. Percentages of antigen-specific splenocytes that stain positivefor interferon-gamma production (IFNγ+) upon stimulation with a pool ofpeptides relevant for the Ag85A antigen (A: CD4+ cells, B: CD8+ cells).

FIG. 14. Percentages of antigen-specific splenocytes that stain positivefor interferon-gamma production (IFNγ+) upon stimulation with a pool ofpeptides relevant for the Ag85B antigen (A: CD4+ cells, B: CD8+ cells).

FIG. 15. Percentages of antigen-specific splenocytes that stain positivefor interferon-gamma production (IFNγ+) upon stimulation with a pool ofpeptides relevant for the TB10.4 antigen (A: CD4+ cells, B: CD8+ cells).

FIG. 16. Overview of percentages of CD4+ and CD8+ splenocytes that stainpositive in ICN, in sera obtained from mice injected with the tripleinserts TB-L (A) and TB-S(B).

FIG. 17. Dose response effect using different doses of TB-S comprising anucleic acid encoding Ag85A, Ag85B and Tb10.4 antigens. CD4 responsetowards Ag85A (A), Ag85B (C) and TB10.4 (E). CD8 response towards Ag85A(B), Ag85B (D) and TB10.4 (F). (G): CD8 response towards Ag85B with theadjusted peptide pool (see example 6): left graph, upon TB-L infection;right graph, upon TB-S infection.

FIG. 18. CD4 and CD8 responses after priming with BCG and boosting withdifferent Ad-TB vectors. CD4 response towards Ag85A (A), Ag85B (C) andTB10.4 (E). CD8 response towards Ag85A (B), Ag85B (D) and TB10.4 (F).

FIG. 19. Nucleotide sequence of TB-LM

FIG. 20. Nucleotide sequence of TB-SM

FIG. 21. Nucleotide sequence of TB-FLM

FIG. 22. Amino acid sequence of TB-LM

FIG. 23. Amino acid sequence of TB-SM

FIG. 24. Amino acid sequence of TB-FLM

FIG. 25. Ag85A stimulation in a BCG prime/Ad35-TB boost experiment witha long-term read-out. Upper panel: CD4 response, lower panel: CD8response. Ad35.E=empty Ad35 virus.

FIG. 26. Ag85B stimulation in a BCG prime/Ad35-TB boost experiment witha long-term read-out. Upper panel: CD4 response, lower panel: CD8response. Ad35.E=empty Ad35 virus.

FIG. 27. TB10.4 stimulation in a BCG prime/Ad35-TB boost experiment witha long-term read-out. Upper panel: CD4 response, lower panel: CD8response. Ad35.E=empty Ad35 virus.

SUMMARY OF THE INVENTION

The present invention relates to recombinant viral vectors, preferablyreplication defective adenoviruses, more preferably recombinant humanadenovirus serotypes Ad11, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50,wherein the viral vectors comprise a heterologous nucleic acid sequenceencoding for (fusion) polypeptides of at least two antigens from one ormore tuberculosis causing bacilli. The encoded antigens may be directlylinked, i.e. forming one single polypeptide. In one preferredembodiment, the antigens are present in a precursor polyprotein, in thesense that they are connected via a linker sequence recognized by aspecific protease that is co-expressed. The heterologous nucleic acidmay comprise the gene encoding the protease. The fusion proteins withthe direct linkages elicit desired immune responses due to the antigenspresent in the fusion product, whereas the proteins comprising theprotease sites are cleaved into separate discrete antigen forms, eachcontributing to the desired immune response. The protease is preferablylinked to the antigens by a protease recognition-site recognized by acellular protease. Both set-ups provide additional or even synergisticeffects in comparison to vaccination or therapy in which viral vectorsare used that comprise only a single transgene-encoding unit. Moregenerally, the invention also relates to viral vectors comprising aheterologous nucleic acid sequence encoding multiple antigens separatedby protease specific cleavage sites. It is to be understood that suchantigens may be from a wide variety of sources including, but notlimited to, infectious agents such as viruses, bacteria and parasites,and are thus according to this aspect of the invention not limited toantigens from tuberculosis-causing bacilli. The antigens fromTuberculosis mycobacterium serve as non-limiting examples of how suchmultivalent viral vector vaccines are generated and how, upon entry intothe host cell, the antigens are separated, and they are able tocontribute to the immune response.

The invention also relates to the use of genetic adjuvants that areco-expressed from the viral vector. These adjuvants are encoded by anucleic acid, which is part of the heterologous nucleic acid sequenceintroduced into the viral vector genome. The adjuvant is expressedtogether with the specific antigen(s) and is thereby able to stimulatethe immune response towards the antigen(s). Clearly, the sequenceencoding the adjuvant may be linked directly to the sequence encodingthe antigen(s), but is preferably separated from the sequence encodingthe one or more antigen(s) by the linker sequence encoding the proteaserecognition site. In the latter case, the adjuvant is present in thehost separately from the antigen(s) and is able to provide itsimmune-stimulatory effects along with the antigen(s).

DETAILED DESCRIPTION

The present invention relates to multivalent vaccines comprising arecombinant viral vector. A preferred viral vector is a recombinantAdenovirus (Ad) vector. The recombinant adenoviral vector according tothe invention comprises a heterologous nucleic acid sequence encoding atleast two different antigens. The antigens may be within a singlepolypeptide. These determinants may be either antigens from viral,bacterial and parasitic pathogens, or host antigens, such as, but notlimited to, autoimmune antigens or tumor antigens. In a preferredembodiment, the antigens are from tuberculosis (TB)-causing bacilli,more preferable from Mycobacterium tuberculosis, M. africanum or M.bovis or from a combination thereof. The antigens may be the full-lengthnative protein, chimeric fusions between the antigen and a host proteinor mimetic, a fragment or fragments thereof of an antigen thatoriginates from the pathogen, or other mutants that still elicit adesired immune response. Genes encoding TB antigens that may typicallybe used in the viral vectors of the present invention include, but arenot limited to: Ag85A (MPT44), Ag85B (MPT59), Ag85C (MPT45), TB10.4(CFP7), ESAT-6, CFP7A, CFP7B, CFP8A, CFP8B, CFP9, CFP10, CFP10A, CFP11,CFP16, CFP17, CFP19, CFP19A, CFP19B, CFP20, CFP21, CFP22, CFP22A, CFP23,CFP23A, CFP23B, CFP25, CFP25A, CFP26 (MPT51) CFP27, CFP28, CFP29,CFP30A, CFP30B, CWP32, CFP50, MPT63, MTC28, LHP, MPB59, MPB64, MPT64,TB15, TB18, TB21, TB33, TB38, TB54, TB12.5, TB20.6, TB40.8, TB10C,TB15A, TB17, TB24, TB27B, TB13A, TB64, TB11B, TB16, TB16A, TB32, TB32A,TB51, TB14, TB27, HBHA, GroEL, GroES (WO 95/01441, WO 98/44119, U.S.Pat. No. 6,596,281, U.S. Pat. No. 6,641,814, WO 99/04005, WO 00/21983,WO 99/24577), and the antigens disclosed in WO 92/14823, WO 95/14713, WO96/37219, U.S. Pat. No. 5,955,077, U.S. Pat. No. 6,599,510, WO 98/31388,US 2002/0150592, WO 01/04151, WO 01/70991, WO 01/79274, WO 2004/006952,WO 97/09428, WO 97/09429, WO 98/16645, WO 98/16646, WO 98/53075, WO98/53076, WO 99/42076, WO 99/42118, WO 99/51748, WO 00/39301, WO00/55194, WO 01/23421, WO 01/24820, WO 01/25401, WO 01/62893, WO01/98460, WO 02/098360, WO 03/070187, U.S. Pat. No. 6,290,969, U.S. Pat.No. 6,338,852, U.S. Pat. No. 6,350,456, U.S. Pat. No. 6,458,366, U.S.Pat. No. 6,465,633, U.S. Pat. No. 6,544,522, U.S. Pat. No. 6,555,653,U.S. Pat. No. 6,592,877, U.S. Pat. No. 6,613,881, U.S. Pat. No.6,627,198. Antigen fusions that may be of particular use are thosedisclosed for the first time herein (such as Ag85A-Ag85B-TB10.4 andcombinations thereof), but also known fusions such as ESAT-6-MPT59 andMPT59-ESAT-6 disclosed in WO 98/44119 and in the above-referenceddocuments.

One approach for applying multiple antigens may be by having two or moreseparate expression cassettes present in a single vector, each cassettecomprising a separate gene of interest. This approach clearly hasdisadvantages, for instance related to space availability in the vector:separate cassettes generally comprise separate promoters and/or inducersand separate polyadenylation signal sequences. Such cassettes typicallyrequire separate positions in the viral vector, resulting in morelaborious cloning procedures, whereas a phenomenon known as ‘promoterinterference’ or ‘squelching’ (limited availability of cellular factorsrequired by the promoters to act) may restrict the expression levelsfrom the different promoters.

As exemplified by the recombinant viral vectors disclosed hereinrelating to fusions between multiple TB antigens, one is now able tomake recombinant adenoviral vectors comprising several nucleic acidsencoding more than one antigen, which viral vector elicits a strongimmune response, whereas the use of single inserts elicit limitedeffects. Clearly, these vectors encode recombinant genetic chimeras,which express the two or more antigens in a single cistronic mRNA, forexample in the form of a fusion protein. This approach can be effectivewhen DNA vaccines or the viral vectors are being used to invoke T cellimmunity to the passenger antigens. However, such fusion proteins mayhave additional drawbacks that cannot always be envisioned beforehand,as it was found that such fusions might skew immunodominant patterns anddo not always invoke immunity to all target antigens with equal potency,whereas a second and perhaps more significant drawback to expression ofgenetic fusions is that the individual components may not fold to anative conformation, due to the close presence of their fusion partneror other reasons. As a result of this, genetic fusions may invokeantibody responses to nonsense epitopes and such antibodies do notrecognize native epitopes displayed by the founder pathogens and may bepoor at combating infection.

The inventors of the present invention have now developed a systemwherein multiple antigens are encoded by a single heterologous nucleicacid sequence, wherein the expressed polyprotein is processed into thediscrete antigenic polypeptides. Thus, in one embodiment, the presentinvention relates to viral vectors that enable the expression ofmultiple antigens that are subsequently processed into the discreteantigens thus avoiding the possible limitations associated with geneticfusions, while also excluding the need for separate expressioncassettes. Heretofore, no compositions or methods have been describedthat enable precise processing of viral vector-expressed genetic fusionsinto discrete antigens. The expression of multiple antigens encoded bynucleic acids comprised in a DNA or viral vector, which antigens aresubsequently processed into discrete antigens is demonstrated by the useof a protease (PR), such as the viral protease encoded by Avian LeucosisVirus (ALV; referred to as PR-ALV herein). In ALV, ALV-PR forms theC-terminal domain of the gag protein, which is known to catalyse theprocessing of gag and gag-pol precursors, a critical step during ALVreplication (reviewed by Skalka. 1989).

A unique ALV-PR directed-processing system was created. A polyproteincontaining ALV-PR and given antigens is expressed by DNA or viralvectors, in which ALV-PR preferably forms the N-terminus of apolyprotein followed by antigen sequences that are linked with ALV-PRdigestion sites. Two different cleavage sites are preferably used in thesystem. One cleavage site (GSSGPWPAPEPPAVSLAMTMEHRDRPLV; SEQ ID NO:22)is to release ALV-PR and the other cleavage site(PPSKSKKGGAAAMSSAIQPLVMAVVNRERDGQTG; SEQ ID NO:21) is recognized byALV-PR and used to separate the other encoded antigens in discretepolypeptides.

Alternatively, the PR and its cleavage sites may be encoded by or basedon other retroviruses such as Human Immunodeficiency Virus (HIV), murineleukaemia virus, Simian Immunodeficiency Virus (SIV) and Rous SarcomaVirus.

According to a preferred embodiment, the invention discloses recombinantviral vectors comprising nucleic acid sequences encoding multipleantigens from Mycobacterium tuberculosis, wherein the different nucleicacid sequences are separated from each other by sequences encoding theALV protease recognition site. In this the discrete TB antigens areproduced as a polyprotein and subsequently processed such that they arecleaved into discrete antigenic polypeptides, each contributing to theimmune response. It is to be understood that the ALV protease system isnot to be limited to the use of TB-specific antigens. A person skilledin the art will appreciate the possibility that the system has forapplying other antigens, different from or in combination with TBantigens, and its applicability in other therapeutic settings such asgene therapy and tumor vaccination.

Preferably, the viral vector comprising the multiple antigen-encodingsequences separated by protease sites is an adenoviral vector. The viralvector may be the viral particle itself, whereas the term viral vectoralso refers to the nucleic acid encoding the viral particle. Theadenoviral vector is preferably a recombinant vector based on, orderived from, an adenovirus species or serotype that encountersneutralizing activity in a low percentage of the target population. Suchadenoviruses are also sometimes referred to as ‘rare’ adenoviruses asthey generally do not regularly circulate within the human population.Preferred serotypes are therefore Ad11, Ad24, Ad26, Ad34, Ad35, Ad48,Ad49 and Ad50.

As used herein, “antigen” means a protein or fragment thereof, whichwhen expressed in an animal or human cell or tissue is capable oftriggering an immune response. Examples include but are not limited to,viral proteins, bacterial proteins, parasite proteins, cytokines,chemokines, immunoregulatory agents, and therapeutic agents. The antigenmay be a wild type protein, a truncated form of that protein, a mutatedform of that protein or any other variant of that protein, in each casecapable of contributing to immune responses upon expression in theanimal or human host to be vaccinated. It is to be understood that whenantigens are directly fused, this fusion is the result of recombinantmolecular biology; thus, a direct fusion of two antigens as used hereindoes not refer to two antigenic parts of a single wild type protein asit occurs in nature. For the sake of clarity, when two antigenic partsof a single wild type protein (which two parts are normally directlylinked within the protein) are linked via linkers as disclosed herein(such as through the ALV protease site, as discussed below), such fusionis part of the present invention. In preferred embodiments, the presentinvention relates to different proteins (having antigenic activity) thatare either directly linked or that are linked through one or moreprotease sites. In a more preferred embodiment, the gene encoding theprotease is linked to the protein(s) of interest, even more preferablythrough yet another protease site.

The different antigens are not necessarily from one pathogenic species.Combinations of different antigens from multiple species, wherein thedifferent antigens are encoded by nucleic acid sequences within a singlevector are also encompassed by the present invention.

A “host antigen” means a protein or part thereof that is present in therecipient animal cell or tissue, such as, but not limited to, a cellularprotein, an immunoregulatory agent, or a therapeutic agent.

The antigen may be encoded by a codon-optimized, synthetic gene and maybe constructed using conventional recombinant DNA methods.

As mentioned, the antigen that is expressed by the recombinant viralvector comprising the ALV protease system can be any molecule that isexpressed by any viral, bacterial, or parasitic pathogen prior to orduring entry into, colonization of, or replication in their animal host.These pathogens can be infectious in humans, domestic animals or wildanimal hosts.

The viral pathogens, from which the viral antigens are derived, include,but are not limited to: Orthomyxoviruses, such as influenza virus;Retroviruses, such as RSV, HTLV-1, and HTLV-II, Herpesviruses such asEBV; CMV or herpes simplex virus; Lentiviruses, such as HIV-1 and HIV-2;Rhabdoviruses, such as rabies virus; Picornaviruses, such as Poliovirus;Poxviruses, such as vaccinia virus; Rotavirus; and Parvoviruses, such asAdeno-Associated Viruses (AAV).

Examples of viral antigens can be found in the group including but notlimited to the Human Immunodeficiency Virus (HIV) antigens Rev, Pol,Nef, Gag, Env, Tat, mutant derivatives of Tat, such as Tat-Δ31-45, T-and B-cell epitopes of gp120, chimeric derivatives of HIV-1 Env andgp120, such as a fusion between gp120 and CD4, a truncated or modifiedHIV-1 Env, such as gp140 or derivatives of HIV-1 Env and/or gp140. Otherexamples are the hepatitis B surface antigen, rotavirus antigens, suchas VP4 and VP7, influenza virus antigens such as hemagglutinin,neuraminidase, or nucleoprotein, and herpes simplex virus antigens suchas thymidine kinase.

Examples of bacterial pathogens, from which the bacterial antigens maybe derived, include but are not limited to, Mycobacterium spp.,Helicobacter pylori, Salmonella spp., Shigella spp., E. coli, Rickettsiaspp., Listeria spp., Legionella pneumoniae, Fansicella spp., Pseudomonasspp., Vibrio spp., and Borellia burgdorferi.

Examples of protective antigens of bacterial pathogens include thesomatic antigens of enterotoxigenic E. coli, such as the CFA/I fimbrialantigen and the nontoxic B-subunit of the heat-labile toxin; pertactinof Bordetella pertussis, adenylate cyclase-hemolysin of B. pertussis,fragment C of tetanus toxin of Clostridium tetani, OspA of Borelliaburgdorferi, protective paracrystalline-surface-layer proteins ofRickettsia prowazekii and Rickettsia typhi, the listeriolysin (alsoknown as “Llo” and “Hly”) and/or the superoxide dismutase (also know as“SOD” and “p60”) of Listeria monocytogenes, urease of Helicobacterpylori, and the receptor-binding domain of lethal toxin and/or theprotective antigen of Bacillus anthrax.

The parasitic pathogens, from which the parasitic antigens are derived,include but are not limited to: Plasmodium spp. such as Plasmodiumfalciparum, Trypanosome spp. such as Trypanosoma cruzi, Giardia spp.such as Giardia intestinalis, Boophilus spp., Babesia spp. such asBabesia microti, Entamoeba spp. such as Entamoeba histolytica, Eimeriaspp. such as Eimeria maxima, Leishmania spp., Schistosome spp., Brugiaspp., Fascida spp., Dirofilaria spp., Wuchereria spp., and Onchocereaspp.

Examples of protective antigens of parasitic pathogens include thecircumsporozoite (CS) or Liver Stage Specific (LSA) antigens LSA-1 andLSA-3 of Plasmodium spp. such as those of P. bergerii or P. falciparum,or immunogenic mutants thereof; the merozoite surface antigen ofPlasmodium spp., the galactose specific lectin of Entamoeba histolytica,gp63 of Leishmania spp., gp46 of Leishmania major, paramyosin of Brugiamalayi, the triose-phosphate isomerase of Schistosoma mansoni, thesecreted globin-like protein of Trichostrongylus colubriformis, theglutathione-S-transferase of Frasciola hepatica, Schistosoma bovis andS. japonicum, and KLH of Schistosoma bovis and S. japonicum.

As mentioned earlier, the recombinant viral vectors comprising nucleicacids encoding the ALV or ALV-like protease may encode host antigens,which may be any cellular protein, immunoregulatory agent, ortherapeutic agent, or parts thereof, that may be expressed in therecipient cell, including but not limited to tumor, transplantation, andautoimmune antigens, or fragments and derivatives of tumor,transplantation, and autoimmune antigens thereof. Thus, in the presentinvention, viral vectors may encode tumor, transplant, or autoimmuneantigens, or parts or derivatives thereof. Alternatively, the viralvectors may encode synthetic genes (made as described above), whichencode tumor-specific, transplant, or autoimmune antigens or partsthereof. Examples of such antigens include, but are not limited to,prostate specific antigen, MUC1, gp100, HER2, TAG-72, CEA, MAGE-1,tyrosinase, CD3, and TAS beta chain.

Clearly, the ALV protease site technology as disclosed herein is alsoapplicable for gene therapy applications by introducing multiplepolypeptides in a single polyprotein and having the polyproteinprocessed into discrete polypeptides in hosts in need of these multiple(discrete) polypeptides.

As a means to further enhance the immunogenicity of the viral vectors,expression cassettes are constructed that encode at least one antigenand an adjuvant, and can be used to increase host responses to theantigen expressed by said viral vectors. Such adjuvants are herein alsoreferred to as ‘genetic adjuvants’ as genes encode the proteins that actas adjuvant. A preferred use is made of the protease and the linkingprotease sites as described above to have the antigen cleaved from theadjuvant after translation, although in certain embodiments the adjuvantmay also be directly linked to the antigen.

The particular adjuvant encoded by the viral vectors may be selectedfrom a wide variety of genetic adjuvants. In a preferred embodiment, theadjuvant is the A subunit of cholera toxin (CtxA; examples: GenBankaccession no. X00171, AF175708, D30053, D30052), or functional partsand/or functional mutant derivatives thereof, such as the A1 domain ofthe A subunit of Ctx (CtxA1; GenBank accession no. K02679).Alternatively, any bacterial toxin that is a member of the family ofbacterial adenosine diphosphate-ribosylating exotoxins may be used.Non-limiting examples are the A subunit of heat-labile toxin (EltA) ofenterotoxigenic E. coli, and the pertussis toxin S1 subunit. Otherexamples are the adenylate cyclase-hemolysins such as the cyaA genes ofBordetella pertussis, B. bronchiseptica or B. parapertussis.Alternatively, the particular ADP-ribosyltransferase toxin may be anyderivative of the A subunit of cholera toxin (i.e. CtxA), or partsthereof (i.e. the A1 domain of the A subunit of Ctx (i.e. CtxA1), fromany classical Vibrio cholerae strain (e.g. strain 395) or El Tor V.cholerae (e.g. strain 2125) that display reduced ADP-ribosyltransferasecatalytic activity but retain the structural integrity, including butnot restricted to replacement of arginine-7 with lysine (R7K), serine-41with phenylalanine (S41F) serine-61 with lysine (S61K), serine-63 withlysine (S63K), valine-53 with aspartic acid (V53D), valine-97 withlysine (V97K) or tyrosine-104 with lysine (Y104K), or combinationsthereof. Alternatively, the particular ADP-ribosyltransferase toxin maybe any derivative of cholera toxin that fully assemble, but are nontoxicproteins due to mutations in the catalytic-site, or adjacent to thecatalytic site, respectively. Such mutants are made by conventionalsite-directed mutagenesis procedures, as described below.

In another embodiment, the ADP-ribosyltransferase toxin is anyderivative of the A subunit of heat-labile toxin (LtxA) ofenterotoxigenic Escherichia coli isolated from any enterotoxigenic E.coli, including but not restricted to E. coli strain H10407 that displayreduced ADP-ribosyltransferase catalytic activity but retain thestructural integrity, including but not restricted to R7K, S41F, S61K,S63K, V53D, V97K or Y104K, or combinations thereof. Alternatively, theparticular ADP-ribosyltransferase toxin may be any fully assembledderivative of cholera toxin that is nontoxic due to mutations in, oradjacent to, the catalytic site. Such mutants are made by conventionalsite-directed mutagenesis procedures, as described below.

Although ADP-ribosylating toxins are potent adjuvants, the adjuvantencoded by the viral vectors in the present invention may also be anybioactive protein from viral, bacterial or protozoan organisms,immunoregulatory DNA, double stranded RNA or small inhibitory RNA(herein referred to as siRNA). The particular bioactive protein can beselected from but not restricted to the following classes:

Class 1. This class of adjuvants induce apoptosis by inhibiting Rho, ahost small GTPase. Inhibition of Rho has been clearly associated withthe induction of apoptosis. Induction of apoptosis is useful method todrive bystander T cell responses and a potent method for the inductionof CTLs. This strategy has not been evaluated in any experimental systemthus far. The active domain of SopE is contained in amino acids 78-240and it only requires a 486 bp gene for expression. The catalytic domainof E. coli CNF-1 is likely to possess similar properties.Class 2. Bacterial porins have been shown to possess immune-modulatingactivity. These hydrophobic homotrimeric proteins form pores that allowpassage of molecules of Mr<600 Da through membranes. Examples of porinsinclude the OmpF, OmpC and OmpD proteins of the Enterobacteriaceae.Class 3. Double stranded RNA (dsRNA) activates host cells, includingdendritic cells. Expression of an mRNA that encodes an inverted repeatspaced by an intro or ribozyme will result in the expression of dsRNA.Class 4. The peptide motive [WYF]xx[QD]xx[WYF] is known to induceCD1d-restricted NK T cell responses (Kronenberg and Gapin, 2002).Expression of a peptide with this motif fused to a T4 fibritincoiled-coiled motif will produce trimeric peptide that will cross-linkTCRs on CD1d-restricted T cells, thereby activated innate hostresponses.Class 5. siRNA can be used to target host mRNA molecules that suppressimmune responses (e.g. kir), regulate immune responses (e.g. B7.2) orprevent cross presentation (e.g. Rho).Class 6. siRNA's can be used for vaccines that target costimulatorymolecules, such as CD80 and CD86. Inhibition of these molecules willprevent co-stimulation thereby resulting in T cell anergy.

Surprisingly, as disclosed herein, it has been found that antigens fromTB-causing bacilli, such as the TB10.4 protein may not only act as anantigen in itself, but even act as an adjuvant towards other TBantigens, for instance Ag85A: In the case where a triple insert waspresent (Ag85A-Ag85B-TB10.4) it was surprisingly found that the presenceof TB10.4 in this construct stimulated the immune response towardsAg85A, whereas the absence of TB10.4 revealed a minor effect towardsCD8+ splenocytes (see example 4 and FIG. 13B). The adjuvant effect ofTB10.4 was further investigated and it was found that TB10.4 indeedstimulated the activation of CD8 cells directed against Ag85A whenpresent in a triple construct, whereas an infection with separatevectors each encoding the separate antigens did not result in suchstimulation, strongly suggesting that the TB10.4 antigen should bepresent, either in the same vector, or present within the sametranslated product.

The present invention also relates to viral vectors that encode at leastone antigen and a cytokine fused by a protease recognition site. Suchvectors are used to increase host responses to the passenger antigen(s)expressed by said viral vectors. Examples of cytokines encoded by theviral vectors are interleukin-4 (IL-4), IL-5, IL-6, IL-10, IL-12_(p40),IL-12_(p70), TGFβ, and TNFα.

Recombinant DNA and RNA procedures for the introduction of functionalexpression cassettes to generate viral vectors capable of expressing animmunoregulatory agent in eukaryotic cells or tissues are known in theart.

Herein, compositions and methods are described for the construction ofviral vectors that express more than one antigen from a TB-causingbacillus, preferably Mycobacterium tuberculosis, M. africanum and/or M.bovis. Preferably, the viral vector is a replication-defectiverecombinant adenoviral vector. One extensively studied and generallyapplied adenovirus serotype is adenovirus 5 (Ad5). The existence ofanti-Ad5 immunity has been shown to suppress substantially theimmunogenicity of Ad5-based vaccines in studies in mice and rhesusmonkeys. Early data from phase-1 clinical trials show that this problemmay also occur in humans.

One promising strategy to circumvent the existence of pre-existingimmunity in individuals previously infected with the most common humanadenoviruses (such as Ad5), involves the development of recombinantvectors from adenovirus serotypes that do not encounter suchpre-existing immunities. Human adenoviral vectors that were identifiedto be particularly useful are based on serotypes 11, 26, 34, 35, 48, 49,and 50 as was shown in WO 00/70071, WO 02/40665 and WO 2004/037294 (seealso Vogels et al. 2003). Others have found that also adenovirus 24(Ad24) is of particular interest as it is shown to be a rare serotype(WO 2004/083418). In a preferred embodiment said viral vector is thus anadenovirus derived from a serotype selected from the group consistingof: Ad11, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50. The advantage ofthis selection of human adenoviruses as vaccine vectors lies clearly inthe fact that humans are not regularly infected with these wild typeadenoviruses. As a consequence, neutralizing antibodies against theseserotypes is less prevalent in the human population at large. This is incontrast to serotype 5, because humans are quite regularly infected withthis wild type serotype. The immune responses raised during an infectionwith a parental wild-type serotype can negatively impact the efficacy ofthe recombinant adenovirus serotype when used as a subsequentrecombinant vaccine vector, such as a vaccine against malaria in whichadenoviruses are applied. The spread of the different adenovirusserotypes in the human worldwide population differs from one geographicarea to the other. Generally, the preferred serotypes encounter a lowneutralizing activity in hosts in most parts of the world, as outlinedin WO 00/70071. In another preferred embodiment, the adenovirus is asimian, canine or a bovine adenovirus, since these viruses also do notencounter pre-existing immunity in the (human) host to which therecombinant virus is to be administered. The applicability of simianadenoviruses for use in human gene therapy or vaccines is wellappreciated by those of ordinary skill in the art. Besides this, canineand bovine adenoviruses were found to infect human cells in vitro andare therefore also applicable for human use. Particularly preferredsimian adenoviruses are those isolated from chimpanzee. Examples thatare suitable include C68 (also known as Pan 9; U.S. Pat. No. 6,083,716)and Pan 5, 6 and 7 (WO 03/046124); See also WO 03/000851.

Thus, choice of the recombinant vector is influenced by those thatencounter neutralizing activity in a low percentage of the humanpopulation in need of the vaccination. The advantages of the presentinvention are multi-fold. Recombinant viruses, such as recombinantadenoviruses, can be produced to very high titers using cells that areconsidered safe, and that can grow in suspension to very high volumes,using medium that does not contain any animal- or human derivedcomponents. Also, it is known that recombinant adenoviruses elicit adramatic immune response against the protein encoded by the heterologousnucleic acid sequence in the adenoviral genome. The inventors of thepresent invention realized that a vaccine comprising multiple antigenswould provide a stronger and broader immune response towards theTB-causing bacillus. Moreover, despite the fact that a single antigencould by itself induce protection in inbred strains of mice, a cocktailcomprising several antigens is conceivably a better vaccine forapplications in humans as it is less likely to suffer from MHC relatedunresponsiveness in a heterogeneous population.

However, from a practical standpoint of vaccine development, a vaccineconsisting of multiple constructs would be very expensive to manufactureand formulate. In addition to simplifying the manufacturing process, asingle construct may ensure equivalent uptake of the components byantigen presenting cells and in turn, generate an immune response thatis broadly specific.

In one particular aspect of the invention the replication-defectiverecombinant viral vector comprises a nucleic acid sequence coding for anantigenic determinant wherein said heterologous nucleic acid sequence iscodon-optimized for elevated expression in a mammal, preferably a human.Codon-optimization is based on the required amino acid content, thegeneral optimal codon usage in the mammal of interest and a number ofaspects that should be avoided to ensure proper expression. Such aspectsmay be splice donor or -acceptor sites, stop codons, Chi-sites, poly(A)stretches, GC- and AT-rich sequences, internal TATA boxes, etcetera.Methods of codon optimization for mammalian hosts are well known to theskilled person and can be found in several places in molecular biologyliterature.

In a preferred embodiment, the invention relates to areplication-defective recombinant adenoviral vector according to theinvention, wherein the adenine plus thymine content in said heterologousnucleic acid, as compared to the cytosine plus guanine content, is lessthan 87%, preferably less than 80%, more preferably less than 59% andmost preferably equal to approximately 45%.

The production of recombinant adenoviral vectors harboring heterolousgenes is well-known in the art and typically involves the use of apackaging cell line, adapter constructs and cosmids and deletion of atleast a functional part of the E1 region from the adenoviral genome (seealso below for packaging systems and preferred cell lines).

The vaccines of the present invention are typically held inpharmaceutically acceptable carriers or excipients. Pharmaceuticallyacceptable carriers or excipients are well known in the art and usedextensively in a wide range of therapeutic products. Preferably,carriers are applied that work well in vaccines. More preferably, thevaccines further comprise an adjuvant. Adjuvants are known in the art tofurther increase the immune response to an applied antigenicdeterminant.

The invention also relates to the use of a kit according to theinvention in the therapeutic, prophylactic or diagnostic treatment ofTB.

The recombinant viral vectors comprising TB antigens of the presentinvention may be used in vaccination settings in which they are appliedin combination with BCG. They may also be applied as a priming agent ora boosting agent, respectively preceding or following a BCG vaccinationto increase the desired immune responses. It can also be envisioned thatdifferent viral vectors as disclosed herein are used in prime-boostsetups, wherein one vector is followed by another. Moreover, vectorscomprising directly linked antigens may be combined as such with vectorscomprising the protease-site linked antigens. Prime-boost settings usingone adenovirus serotype as a prime and another serotype as a boost(selected from the preferred human, simian, canine or bovineadenoviruses) are also envisioned. The viral vectors according to theinvention may also be used in combination with vaccines comprisingpurified (recombinantly produced) antigens and/or with vaccinescomprising naked DNA or RNA encoding similar or the same antigens.

Thus, the invention relates to a recombinant replication-defectiveadenovirus comprising a nucleic acid sequence encoding two or moreantigens from at least one tuberculosis (TB)-causing bacillus. It is tobe understood that a polypeptide may comprise several antigenic parts orantigenic fragments (=antigens). Also, a protein itself may beconsidered as being an ‘antigen’. Preferably, said recombinantadenovirus is a human or a simian adenovirus. More preferably, theadenovirus used as a recombinant vector in the present invention isselected from the group consisting of human adenovirus serotypes Ad11,Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50. The TB-causing bacillusused for providing the preferred antigen(s) is preferably Mycobacteriumtuberculosis, Mycobacterium africanum and/or Mycobacterium bovis, andsaid two or more antigens are preferably selected from the groupconsisting of antigens encoded by the Ag85A, Ag85B, ESAT-6, f72 andTB10.4 open reading frames of M. tuberculosis. In a highly preferredembodiment, said nucleic acid sequence encodes at least two antigensselected from the group consisting of antigens encoded by the Ag85A,Ag85B, and TB10.4 open reading frames of M. tuberculosis. In an evenmore preferred embodiment, the adenovirus according to the inventioncomprises a nucleic acid sequence encoding the full length proteinsAg85A, Ag85B and TB10.4, wherein it is even more preferred that thesethree proteins are encoded by a nucleic acid comprising a sequence inwhich the genes encoding the respective proteins are cloned in that 5′to 3′ order (Ag85A-Ag85B-TB10.4).

The invention relates to a recombinant adenovirus according to theinvention, wherein at least two of said antigens are expressed from onepolyprotein. In one preferred embodiment at least two of said antigensare linked so as to form a fusion protein. The linkage may be direct orvia a connecting linker of at least one amino acid. Where a linker isused to connect two separate antigens and thus to provide a fusionprotein of two or more antigens according to the invention, preferablyone or more linkers according to SEQ ID NO:23 is used.

The invention also relates to a multivalent TB vaccine comprising arecombinant adenovirus according to the invention or a recombinantpolynucleotide vector according to the invention, further comprising apharmaceutically acceptable excipient, and optionally an adjuvant. Manypharmaceutically acceptable recipients and adjuvants are known in theart.

The invention furthermore relates to a method of vaccinating a mammalfor the prevention or treatment of TB, comprising administering to saidmammal a recombinant adenovirus, a multivalent TB vaccine or arecombinant polynucleotide vector according to the invention. In oneaspect, the invention relates to a method of vaccinating a mammal forthe prevention or treatment of TB, comprising the steps of administeringto said mammal a recombinant adenovirus, a multivalent TB vaccine, or arecombinant polynucleotide vector according to the invention as apriming vaccination; and administering to said mammal a recombinantadenovirus, a multivalent TB vaccine, or a recombinant polynucleotidevector according to the invention as a boosting vaccination. Theinvention also relates to a recombinant adenovirus, a multivalent TBvaccine, or a recombinant polynucleotide vector according to theinvention, either one for use as a medicament, preferably in theprophylactic-, therapeutic- or diagnostic treatment of tuberculosis. Theinvention also relates to the use of a recombinant adenovirus, amultivalent TB vaccine, or a recombinant polynucleotide vector accordingto the invention in the preparation of a medicament for theprophylactic-, or therapeutic treatment of tuberculosis.

In one particular aspect, the invention relates to a recombinantpolynucleotide vector comprising a nucleic acid sequence encoding two ormore antigens and a protease-recognition site, wherein said antigens areexpressed as a polyprotein, said polyprotein comprising the proteaserecognition site separating at least two of the two or more antigens.Preferably, said polynucleotide vector is a naked DNA-, a naked RNA-, aplasmid-, or a viral vector. In a preferred embodiment, said viralvector is packaged into a replication-defective human or simianadenovirus. It is to be understood that a viral vector may be seen astwo kinds of entities: the viral DNA encoding the virus may be used as anucleic acid vector, while the virus (comprising the viral vector DNA)may also be used to transfer the nucleic acid of interest to a host cellthrough infection of said host cell. Thus, a ‘vector’ as used hereinrefers to a means for transferring a gene or multiple genes of interestto a host. This may be achieved by direct injections of the DNA, RNA,plasmid, or the viral nucleic acid vector, but may also be achieved byinfecting host cells with a recombinant virus (which then acts as thevector). As exemplified herein, viruses may be used to immunize mammals(for example mice), whereas the DNA (for instance in the form of theadapter plasmid carrying the gene(s) of interest and a part of the viralDNA) may also be directly injected in the mammal for immunizing saidmammal. Vaccines based on naked DNA, or RNA, or plasmids are known inthe art, whereas vaccines based on recombinant viruses are also known.For clarity issues, all entities that deliver a gene or more genes ofinterest to a host cell are regarded as a ‘vector’.

In one preferred embodiment the nucleic acid present in said vectorcomprises a sequence encoding a protease, wherein it is preferred thatsaid protease upon expression is expressed as part of the polyproteinand is linked to at least one of said antigens by a protease-recognitionsite. Particularly preferred protease-recognition sites comprise asequence according to SEQ ID NO:21 or 22. More preferred is arecombinant polynucleotide vector according to the invention, whereinsaid protease is from an Avian Leukosis Virus (ALV). In a preferredaspect, the antigens that are linked through a protease recognition siteare from at least one tuberculosis (TB)-causing bacillus, wherein saidTB-causing bacillus is preferably Mycobacterium tuberculosis,Mycobacterium africanum and/or Mycobacterium bovis. The two or moreantigens are preferably selected from the group consisting of antigensencoded by the Ag85A, Ag85B, ESAT-6, and TB10.4 open reading frames ofM. tuberculosis, wherein said heterologous nucleic acid sequence encodesmost preferably at least two antigens selected from the group consistingof antigens encoded by the Ag85A, Ag85B, and TB10.4 open reading framesof M. tuberculosis. Even more preferred are poluynucleotides accordingto the invention wherein the antigens are the full length Ag85A, Ag85Band TB10.4 polypeptides, of which the encoding genes are cloned in that5′ to 3′ order. Fusion proteins based on these and other tuberculosisantigens were described in U.S. Pat. No. 5,916,558, WO 01/24820, WO03/070187 and WO 2005/061534. However, the use of the nucleic acidsaccording to the present invention, encoding the fusion proteinsdisclosed herein, for incorporation into recombinant adenoviral vectorswas not disclosed.

In yet another aspect, the invention relates to a recombinantpolynucleotide vector comprising a heterologous nucleic acid sequenceencoding an antigen and a genetic adjuvant. The term ‘genetic adjuvant’refers to a proteinaceous molecule that is encoded by a nucleic acidsequence. Said antigen and said genetic adjuvant may be linked directlyor in another embodiment linked indirectly, for instance by a connectioncomprising a first protease-recognition site. In another preferredaspect, said polynucleotide vector is a naked DNA-, a naked RNA-, aplasmid-, or a viral vector. The viral vector is preferably packagedinto a replication-defective human or simian adenovirus, wherein saidadenovirus is even more preferably selected from the group consisting ofhuman adenovirus serotypes Ad11, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 andAd50. Also preferred are nucleic acids comprising a sequence encoding aprotease, wherein said protease is preferably linked to said antigenand/or to said genetic adjuvant by a second protease-recognition site.The preferred second protease-recognition site comprises a sequenceaccording to SEQ ID NO:22, whereas the preferred firstprotease-recognition site comprises a sequence according to SEQ IDNO:21. A preferred protease is a protease from an Avian Leukosis Virus(ALV), while the antigens are preferably from at least a tuberculosis(TB)-causing bacillus, more preferably Mycobacterium tuberculosis,Mycobacterium africanum and/or Mycobacterium bovis. Preferred antigensare selected from the group consisting of: Ag85A, Ag85B, ESAT-6 andTB10.4. A most preferred embodiment is a vector wherein saidheterologous nucleic acid sequence encodes at least two antigensselected from the group consisting of M. tuberculosis antigens Ag85A,Ag85B, and TB10.4, wherein it is further preferred to have a fusionpolypeptide comprising the full length Ag85A, Ag85B and TB10.4 proteins,in that order from N- to C-terminus.

As disclosed herein, the TB10.4 has unexpected adjuvant activity, as itwas found that it stimulates the immune response towards the other(especially Ag85A) antigen present in the polyprotein. The TB10.4adjuvant is a preferred genetic adjuvant. Thus, the invention alsoprovides a recombinant vector comprising a nucleic acid encoding theTB10.4 antigen with at least one other antigen, which antigen ispreferably a tuberculosis antigen, more preferably the Ag85A antigen. Inan even more preferred embodiment, the vector comprises a nucleic acidencoding the TB10.4 antigen and at least the Ag85A and Ag85B antigens.As outlined below, the TB10.4 is suggested to increase the processing ofthe multiple-antigen translation product towards the proteosome,resulting in a highly significant increase in CD8 response. It is verylikely that the effect is not limited to Ag85A and TB10.4 alone, with awider applicability of the TB10.4 antigen than limited to tuberculosisvaccines alone. Thus, the invention, in yet another embodiment, alsorelates to a recombinant vector comprising a nucleic acid encodingTB10.4 and at least one other antigen, wherein the other antigen is nota Mycobacterium antigen. The invention discloses the use of theMycobacterium TB10.4 antigen as a genetic adjuvant. Moreover, theinvention discloses the use of the TB10.4 antigen in the manufacture ofa medicament for the treatment, diagnosis and/or prophylaxis of adisease other than tuberculosis, and at least in a disease in which theimmune response towards the antigen of interest needs to be stimulatedby the action of an adjuvant. So, it is disclosed that an antigen withinTB10.4 of Mycobacterium tuberculosis can act as an adjuvant towardsother antigens, such as Ag85A. Thus, the invention also relates to theuse of the Mycobacterium antigen TB10.4 as a genetic adjuvant.Furthermore, in another embodiment, the invention relates to the use ofthe Mycobacterium antigen TB10.4 in the preparation of a medicament forthe treatment or prophylaxis of a disease in which the immune responseof a host towards a certain antigen or therapeutic component of interestneeds to be stimulated. The skilled person would be able to determinethe level of immune response towards a given antigen of interest andwhether an extra stimulation by the use of an adjuvant, such as agenetic adjuvant, herein exemplified by TB10.4, might be beneficial in atreated subject.

The invention further relates to a recombinant polynucleotide vectoraccording to the invention, wherein said genetic adjuvant comprises acholera toxin (CtxA1) or a mutant derivative thereof, said mutantderivative comprising a serine to lysine substitution at amino acidposition 63 (A1_(K63)). The invention also relates to a multivalent TBvaccine comprising a recombinant polynucleotide vector according to theinvention, further comprising a pharmaceutically acceptable excipient,and optionally an adjuvant.

EXAMPLES Example 1 Construction of Ad35-Based Adapter Plasmids CarryingM. tuberculosis Antigens

Here, the construction of adapter plasmids suitable to generateE1-deleted Ad35-based vectors capable of expressing single or multipleTB antigens is described. The examples relate to TB antigens Ag85A(Swissprot #P17944), Ag85B (Swissprot #P31952) and TB10.4 (Swissprot#O53693) as non-limiting examples of means and methods to generatesingle- and multi-antigen vaccine preparations using Adenovirus-basedreplication-defective vectors. As already stated above, the principlesapplied here can also be applied to any combination of prophylactic ortherapeutic polypeptide.

Construction of pAdApt35Bsu.myc.

Adapter plasmid pAdApt35Bsu is described in applicant's application WO2004/001032. This plasmid contains the left part of the Ad35 genome(including the left Inverted Terminal Repeat (ITR)), further lacking afunctional E1 region, and an expression cassette comprising a CMVpromoter inserted into the E1 region. The adapter also comprises afunctional pIX promoter and a region of Ad35 downstream of the E1region, which region is sufficient for a homologous recombination eventwith a cosmid comprising the remaining part of the Ad35 genome, leadingto the generation of a recombinant replication-defective adenovirus in apackaging cell, which packaging cell provides all necessary elements andfunctions for a functional replication and packaging of the virus to beproduced. The generation of recombinant adenoviruses using such adapterplasmids is a process well known to the skilled person.

pAdApt35Bsu was digested with NheI and XbaI and the 5 kbvector-containing fragment was isolated from agarose gel using theQiaquick gel extraction kit (Qiagen) according to manufacturer'sinstructions. A double-stranded (ds) linker was prepared from thefollowing single stranded (ss) oligos (synthesized by Sigma): Myc-oligo1: 5′-CTA GCA AGA AAA CCG AGC AGA AGC TGA TCT CCG AGG AGG ACC TGT GATAAT-3′ (SEQ ID NO:1) and Myc-oligo 2: 5′-CTA GAT TAT CAC AGG TCC TCC TCGGAG ATC AGC TTC TGC TCG GTT TTC TTG-3′ (SEQ ID NO:2). The twooligonucleotides were mixed using 2 μl of 0.5 μg/μl stocks in a totalvolume of 20 μl annealing buffer (10 mM Tris-HCl pH 7.9, 10 mM MgCl₂, 1mM Dithiotreitol), incubated at 98° C. for 2 min and subsequently cooleddown to 4° C. at a rate of 0.6° C. per min using a PCR machine. Theresulting ds linker was then ligated with the above prepared pAdApt35Bsuvector in 3×, 6× or 9× molar excess of the linker. Colonies were testedfor insertion of the linker sequence in correct orientation by digestionwith NheI or XbaI, sites that are restored only in correct orientation.Sequencing confirmed that the linker consisted of the expected sequence.The resulting adapter plasmid is named pAdApt35Bsu.myc (FIG. 1).

Below, methods for cloning a large set of different constructs isprovided. An overview of all constructs and their respective inserts isfound in Table I.

TABLE I Construct names and their respective inserts comprising nucleicacids encoding TB antigens. Construct Insert TB-3ALV-dig*-Ag85A-dig-Ag85B TB-3M ALV-dig*-Ag85A-dig-Ag85B-myc TB-4Ag85A-Ag85B TB-4M Ag85A-Ag85B-myc TB-5 Ag85A TB-SM Ag85A-myc TB-6 Ag85BTB-6M Ag85B-myc TB-7 TB10.4 TB-7M TB10.4-myc TB-S Ag85A-Ag85B-TB10.4TB-SM Ag85A-Ag85B-TB10.4-myc TB-L ALV-dig*-Ag85A-dig-Ag85B-dig-TB10.4TB-LM ALV-dig*-Ag85A-dig-Ag85B-dig-TB10.4-myc TB-FLAg85A-X-Ag85B-X-TB10.4 TB-FLM Ag85A-X-Ag85B-X-TB10.4-myc ALV = AvianLeukosis Virus protease; dig* = a protease recognition site recognizedby a cellular protease; dig = protease recognition site recognized bythe ALV protease; X = flexible linker, not being a protease recognitionsite; myc = myc-tag.Construction of pAdApt35Bsu-Based Adapter Plasmids Containing Three TBAntigens

The heterologous nucleic acids of the present invention encode the threeM. tuberculosis antigens Ag85A, Ag85B and TB10.4 as a poly-protein fromone mRNA. All fusion sequences indicated with an ‘M’ contain a mycepitope (myc-tag: SKKTEQKLISEEDL; SEQ ID NO:9) attached to the 3′ end ofthe sequence to allow analysis of expression using myc-specificantibodies in case the antibodies specific for the separate TB antigensdo not recognize the fusions properly. Thus, the ‘M’ in the names of allconstructs described below relates to the myc-tag, whereas also allconstructs were made without a myc-tag.

In a first embodiment (TB-SM) the three antigens are expressed as adirect fusion polyprotein: Ag85A-Ag85B-TB10.4-myc(TB-S=Ag85A-Ag85B-TB10.4).

In a second embodiment (TB-LM) the polyprotein precursor contains aprotease, which cleaves the three antigens intra-cellularly onincorporated digestion sites that separate them: linker/digestion sitesequence: PPSKSKKGGAAAMSSAIQPL VMAVVNRERDGQTG (SEQ ID NO:21). Thisdigestion occurs through a sequence-specific protease fused to theN-terminus of the fusion protein. This protease, derived from the gaggene of the Avian Leukosis Virus (ALV) is also cleaved resulting in fourseparate proteins after protease digestion. The polyprotein may be asfollows: ALV-dig*-Ag85A-dig-Ag85B-dig-TB10.4-myc (in which ‘dig*’relates to the digestion site separating the protease from the antigens[GSSGPWPAPEPPAV SLAMTMEHRDRPLV; SEQ ID NO:22] of the protease and ‘dig’relates to the digestion site between the antigens, see above;TB-L=ALV-dig*-Ag85A-dig-Ag85B-dig-TB10.4). Both protease-cleavablelinkers as well as self-cleavage linkers may be used in the vectors ofthe present invention and are encompassed herein. The use ofself-processing cleavage sites has been described in WO 2005/017149.

In a third embodiment the poly-protein (TB-FLM) comprises the mentionedM. tuberculosis antigens separated by a linker sequence that is notcleaved (as in the second embodiment described above) but allows properand independent folding of each of the three antigens:Ag85A-X-Ag85B-X-TB10.4-myc (in which ‘X’ relates to a flexible linker:GTGGSGGTGSGTGGSV; SEQ ID NO:23). All these fusion proteins were alsomade without the myc-tag (referred to as TB-S, TB-L and TB-FLrespectively) using similar construction methods (see below).

The desired protein sequences were assembled using the above indicatedpublished protein sequences for the M. tuberculosis antigens, the ALVprotease PR p15 sequence as published in Genbank (Acc. No. CAA86524) andthe protease digestion site as in Genbank Acc. No. AAK13202 amino acids476-500. The three protein sequences were then back translated to DNAcoding sequences optimised for expression in humans and subsequentlysynthesized, assembled and cloned in pCR-Script vectors by Geneart(Germany).

The codon-optimized DNA sequences for TB-LM are provided (FIG. 19; SEQID NO:3), TB-SM (FIG. 20; SEQ ID NO:4) and TB-FLM (FIG. 21; SEQ IDNO:5), as well as the protein sequences for TB-LM (FIG. 22; SEQ IDNO:6), TB-SM (FIG. 23; SEQ ID NO:7) and TB-FLM (FIG. 24; SEQ ID NO:8).The myc epitope is contained within the C-terminal sequenceSKKTEQKLISEEDL (SEQ ID NO:9) in each of the fusion proteins, whichsequence is not present in the case of TB-L, TB-S and TB-FL,respectively.

The cloned fusion genes were then digested with HindIII, XbaI and ApaLIafter which the 2.7 kb (TB-L), 2.2 kb (TB-FL) and 2.1 kb (TB-S)fragments were isolated from agarose gel as described above. ApaLIdigestion was done to digest the plasmid vector in fragments that werebetter separable from the inserts. Plasmid pAdApt35Bsu was also digestedwith HindIII and XbaI and the vector-containing fragment was isolatedfrom gel as above. The isolated pAdApt35Bsu vector was ligated inseparate reactions to each of the isolated fragments containing the TBsequences and transformed into DH5α competent bacteria (Invitrogen).Resulting colonies were analysed by digestion with HindIII and XbaI andplasmid clones containing the expected insert were selected. Thisresulted in pAdApt35Bsu.TB.LM (FIG. 2), pAdApt35Bsu.TB.SM (FIG. 3) andpAdApt35Bsu.TB.FLM (FIG. 4), all containing a myc-tag. To generateadapter plasmids expressing the fusion genes without myc-tag the insertsare first PCR-amplified using the following primers and templates:

Fragment TB-L

ALVprot.FW: 5′-GCC CAA GCT TGC CAC CAT GCT GGC CAT GAC CAT GG-3′(SEQ IDNO:10) and 10.4.RE.stop: 5′- GCT AGT CTA GAT TAT CAG CCG CCC CAC TTG GC-3′(SEQ ID NO:11) with TB-LM as template.

Fragment TB-FL and TB-S

85A.FW: 5′-GCC CAA GCT TGC CAC CAT GTT CAG C-3′(SEQ ID NO:12) and10.4.RE.stop with TB-FLM or TB-SM as template.

The amplifications were done with Phusion DNA polymerase (Bioke)according to manufacturer's instructions. The following program wasused: 2 min at 98° C. followed by 30 cycles of (20 sec at 98° C., 30 secat 58° C. and 2 min+30 sec at 72° C.) and ended by 10 min at 72° C. Theresulting fragments were purified using Qiaquick PCR purification kit(Qiagen) and digested with HindIII and XbaI. The digested fragments werethen again purified over a Qiaquick PCR purification column as above andligated with pAdApt35Bsu digested with the same enzymes and purifiedover a Qiaquick PCR purification column. Transformation into competentDH5α bacteria (Invitrogen) and selection of the clones containing thecorrect inserts using HindIII and XbaI as diagnostic enzymes results inpAdApt35Bsu.TB-L, pAdApt35Bsu.TB-S and pAdApt35Bsu.TB-FL. Theseconstructs differ from the constructs presented in FIGS. 2, 3 and 4 inthat they do not contain the myc epitope at the C-terminal end.

Construction of pAdApt35Bsu-Based Adapter Plasmids Containing Two TBAntigens

Here, the construction of adapter plasmids containing two TB antigensusing Ag85A and Ag85B as an example is also described. Obvious to theperson with general skill in the art, other combinations and a differentorder of M. tuberculosis antigens can be made using the general strategyoutlined herein. The fusions that are described here are TB-3M:ALV-dig*-Ag85A-dig-Ag85B-myc and TB-4M: Ag85A-Ag85B-myc, and the sameconstructs without the myc-tag. Specific primers were designed toamplify the Ag85A and Ag85B sequences from the above described TB-LM andTB-SM fusion proteins (see below). Fusions are generated with andwithout (TB-3 and TB-4 respectively) myc-tag as above. Hereto, differentprimer sets and templates are used.

Fragment TB.3M:

ALVprot.FW and 85B.RE myc: 5′- GCC TAG CTA GCG CCG GCT CCC AGG CTG C-3′(SEQ ID NO:13) with TB-LM as template.

Fragment TB.4M:

85A.FW.TB.L: 5′- GCC CAA GCT TGC CAC CAT GTT CAG CAG ACC CGG CCT G-3′(SEQ ID NO:14) and 85B.RE myc (see above) with TBSM as template.

All reactions were done using Phusion (Bioke) DNA polymerase with theconditions as described above. PCR fragments were purified using aQiaquick PCR purification kit, digested with HindIII and NheI and againpurified using the PCR purification kit. The amplified fragments weresubsequently ligated into plasmid pAdApt35Bsu.myc that was digested withthe same enzymes. After transformation into competent DH5α bacteria(Invitrogen) clones were selected that contained an insert of thecorrect length. This resulted in constructs pAdApt35Bsu.TB.3M (FIG. 5)and pAdApt35Bsu.TB.4M (FIG. 6).

Fragments TB.3 and TB.4 were generated using the same forward primersand templates indicated above for fragments TB.3M and TB.4M but using adifferent reverse primer named 85B.RE.stop: 5′-GCT AGT CTA GAT TAT CAGCCG GCT CCC AGG CTG C-3′ (SEQ ID NO:15). Amplified fragments werepurified as above, digested with HindIII and XbaI, and again purified asdescribed intra and cloned into pAdApt35Bsu using HindIII and XbaI ascloning sites. This gave pAdApt35Bsu.TB.3 and pAdApt35Bsu.TB.4 that onlydiffer from the constructs in FIGS. 5 and 6 in that they have nomyc-epitope at the C-terminus.

Other combinations that may be useful but not described in detailedcloning procedures herein are:

ALV-dig*-Ag85B-dig-Ag85A-myc

ALV-dig*-Ag85A-dig-TB10.4-dig-Ag85B-mycALV-dig*-TB10.4-dig-Ag85A-dig-Ag85B-mycALV-dig*-TB10.4-dig-Ag85B-dig-Ag85A-myc

ALV-dig*-Ag85B-dig-Ag85A-dig-TB10.4-myc

ALV-dig*-Ag85B-dig-TB10.4-dig-Ag85A-myc

ALV-dig*-Ag85A-dig-TB10.4-myc ALV-dig*-Ag85B-dig-TB10.4-myc

ALV-dig*-TB10.4-dig-Ag85A-mycALV-dig*-TB10.4-dig-Ag85B-myc

Ag85B-Ag84A-myc Ag85A-TB10.4-myc Ag85B-TB10.4-myc TB10.4-Ag85A-mycTB10.4-Ag85B-myc Ag85A-X-Ag85B-myc Ag85B-X-Ag85A-myc Ag85A-X-TB10.4-mycAg85B-X-TB10.4-myc TB10.4-X-Ag85A-myc TB10.4-X-Ag85B-mycAg85A-X-TB10.4-X-Ag85B-myc Ag85B-X-Ag85A-X-TB10.4-mycAg85B-X-TB10.4-X-Ag85A-myc TB10.4-X-Ag85A-X-Ag85B-mycTB10.4-X-Ag85B-X-Ag85A-myc

Dig*, dig, myc and X all relate to the same features as outlined above.It is to be understood that these constructs may also be producedwithout the myc-tag.Construction of pAdApt35Bsu-Based Adapter Plasmids Containing Single TBAntigens

Here, the Ad35 adapter plasmids containing single TB antigens are alsodescribed. As above, proteins are expressed with or without myc-tag.Hereto, the appropriate coding regions were amplified from the TB-L andTB-S templates using specific primers sets:

Fragment TB.5M

85A.FW.TB.L and 85A.RE myc: 5′-GCC TAG CTA GCG CCC TGG GGG G-3′ (SEQ IDNO:16) using TB-LM as template.

Fragment TB.6M:

85B.FW: 5′- GCC CAA GCT TGC CAC CAT GTT CAG CCG GCC TGG CCT G -3′ (SEQID NO:17) and 85B.RE myc using TB-LM as template.

The fragments without myc-tag were generated with the same forwardprimers but with different reverse primers: 85A.RE.stop (for TB.5):5′-GCT AGT CTA GAT TAT CAG CCC TGG GGG GCA G-3′ (SEQ ID NO:20),85B.RE.stop (for TB.6), and 10.4.RE.stop (for TB.7). All reactions weredone with Phusion (Bioke) as described above. All amplified fragmentswere purified using the Qiaquick PCR purification kit. The fragmentsTB-5M and TB-6M were then digested with HindIII and NheI and afterpurification as described above, cloned into pAdApt35Bsu.myc digestedwith the same enzymes. The fragments TB-7M, TB-5, TB-6 and TB-7 weredigested with HindIII and XbaI. After purification as above fragmentswere ligated into pAdApt35Bsu digested with the indicated restrictionenzymes. This resulted in pAdApt35Bsu.TB.5M (FIG. 7), pAdApt35Bsu.TB.6M(FIG. 8), pAdApt35Bsu.TB.7M (FIG. 9), pAdApt35Bsu.TB.5, pAdApt35Bsu.TB.6and pAdApt35Bsu.TB.7. The latter three differ only from the ones inFIGS. 7, 8 and 9 in that no myc-tag is present at the C-terminus.

Example 2 Generation of Replication-Deficient Ad35 Viruses CarryingNucleic Acids Encoding TB Antigens

Methods to generate stable replication-defective recombinant Ad35-basedadenoviral vectors carrying heterologous expression cassettes are wellknown to the person of skill in the art and were previously described inpublished patent applications WO 00/70071, WO 02/40665, WO 03/104467 andWO 2004/001032. This example describes the generation of Ad35-based TBvectors using PER.C6® cells and Ad35 viruses comprising the Ad5-derivedE4-Orf6 and E4-Orf6/7 genes replacing the homologous E4-Orf6 and 6/7sequences in the Ad35 backbone (generally as described in WO 03/104467and WO 2004/001032). For the purposes of the invention, PER.C6® cellsrefer to cells as deposited on Feb. 29, 1996 as patent deposit under no.96022940 at the European Collection of Cell Cultures (ECACC) at theCentre of Applied Microbiology & Research (CAMR), Porton Down,Salisbury, Wiltshire, SP4 0JG United Kingdom.

The generated adapter plasmids described herein, containing thedifferent TB antigens were digested with Pi-PspI to liberate the Ad35sequences and transgene cassette (adapter fragment) from the plasmidbackbone. Construct pWE.Ad35.pIX-EcoRV (see WO 03/104467 and WO2004/001032) was digested with NotI and EcoRV (fragment 2) and constructpBr.Ad35.AE3.PR5Orf6 (see WO 03/104467 and WO 2004/001032) was digestedwith PacI and NotI (fragment 3). The digested DNA mixes were incubatedat 65° C. to inactivate the enzymes. For each transfection, digestedadapter fragment (360 ng), fragment 2 (1.4 pg) and fragment 3 (1 pg)were mixed to a (maximum) volume of 15 μl and adjusted to 25 μl withDMEM (culture medium, Invitrogen). A second mixture was prepared bymixing 14.4 μl Lipofectamine (Invitrogen) with 10.6 μl DMEM, after whichthe two mixes were added together and mixed by tapping the tube. Theresulting DNA-Lipofectamine mixture was then incubated 30-40 min at roomtemperature after which 4.5 ml DMEM was added to the tube. Duringincubation, PER.C6 cells that were seeded the day before in 6-wellplates at 1.5×10⁶ cells/well in DMEM containing 10% FBS(Invitrogen/GIBCO) and 10 mM MgCl₂, were washed with DMEM. Then, to thefirst two wells 0.5 ml DMEM was added and 0.5 ml of the incubatedtransfection mixture. To the second two wells 0.25 ml medium and 0.75 mlof the transfection mixture was added. The last two wells received 1 mlof the transfection mixture. The 6-well plate was then incubated at 37°C. and 10% CO₂ for 4 h after which an agar overlay was placed asfollows. 30 min before the end of the 4 h incubation period, a mixturecontaining 9 ml 2×MEM (Invitrogen), 0.36 ml FBS, 0.18 ml 1M MgCl₂ and1.3 ml PBS was prepared and placed at 37° C. A sterile pre-made solutionof 2.5% agarose (Seaplaque; Cambrex) in H₂O was melted and also kept at37° C. (at least 15 min prior to use). The transfection medium was thenremoved from the cells and cells were washed with PBS once. Then 7.5 mlof the agar solution was added to the MEM medium mixture, mixed and 3 mlwas quickly added to each well. The overlay was allowed to coagulate inthe flow after which the plates were incubated at 37° C./10% CO₂ for atleast 7 days. When large enough, single plaques were picked from thewells with the lowest number of plaques using pipettes with sterilefilter tips (20 μl). The picked plaques were mixed in 200 μl culturemedium each and 100 μl of this was used to inoculate PER.C6 cells in6-well plates. Upon CPE and after one more amplification of the viruseson PER.C6 cells in T25 flasks cells and medium were harvested andfreeze/thawed once and stored as crude lysates. These virus stocks wereused to confirm the presence of the correct transgene by PCR on isolatedvirus DNA and to test expression. One of the amplified plaques was thenchosen to generate virus seed stocks and to produce batches of purifiedvirus according to procedures known in the art using a two-step CsClpurification method. The concentration of purified viruses was typicallydetermined by HPLC as described by Shabram et al. (1997).

Example 3 Analysis of Expression of TB Antigens Upon Infection with Ad35Viral Vectors

The expression of the fused TB antigens was determined by westernblotting. Hereto, A549 cells were infected with the different Ad35viruses containing the genes encoding the TB antigens. 48 h postinfection, cells were washed twice with PBS (NPBI), lysed and scraped inlysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% DOC, 1% Tween-20in dH₂O supplemented with 1% SDS and Protease inhibitor added as a pill(Roche)). After 5-10 min in lysis buffer on ice, lysates were collectedand cleared by centrifugation. Equal amounts of whole-cell extract werefractionated by using 4-12% Bis-Tris NuPAGE® Pre-Cast Gels (Invitrogen).Proteins were transferred onto Immobilon-P membranes (Millipore) andincubated with a polyclonal antibody directed to the Culture FiltrateProtein of M. tuberculosis. This polyclonal serum was raised in rabbitsagainst an M. tuberculosis culture comprising secreted proteins. Inprinciple the polyclonal serum contains antibodies against Ag85A, Ag85Band TB10.4, which are all secreted proteins. The secondary antibody wasa horseradish-peroxidase-conjugated goat-anti-rabbit antibody (Biorad).The western blotting procedure and incubations were performed accordingto general methods known in the art. The complexes were visualized withthe ECL detection system (Amersham) according to the manufacturer'sprotocol.

FIG. 10A shows the results using Ad35 viruses carrying the TB encodingnucleic acids including the myc epitope as described herein. Thedifferent lanes in FIG. 10A show the different viral vectors used andTable I indicates which name refers to what insert. In the same way,expression of the TB antigens from the Ad35 viruses that do not containa myc epitope was measured (FIG. 10B). FIG. 10C shows a similar result,with the molecular weight indicated on the right hand side. Specific TB(fusion) proteins expressed from Ad35 viruses are detected by thismethod and, in addition, certain cleavage products of TB-3 and TB-L.From FIG. 10A it can be concluded that the polyprotein including allthree TB antigens is expressed, since a higher band in lane TB-LM ispresent as compared to TB-3M (and the band in land TB-S is higher thanthe specific band in TB-4M). Since the TB10.4 is the most C-terminalpolypeptide in the TB-LM and TB-SM polyproteins, this indicates that theentire polyproteins are translated. It is also noted that cleavage isnot complete, although cleavage products can be seen in lanes TB-3M andTB-LM. The Ag85A and Ag85B antigens (lanes TB-5(M) and TB-6(M)respectively) are expressed. No specific staining is found in lanesTB-7(M) related to the TB10.4 antigen. It may be that the antigen is notrecognized in a western blot setting by the CFP polyclonal, whereas itmay also be that the protein has run from the gel or that is poorlyexpressed in A549 cells when present in a single expression construct(TB-7M), while present in a triple construct (as TB-LM, TB-L and TB-S).In FIG. 10A, lane TB-LM a slightly shorter band is visible under thehighest (probably non-cleaved) band. This suggests cleavage of theTB10.4 antigen from the remaining portion of the polyprotein.

Further experiments should reveal the physical presence of the protein,although it is clear that the TB10.4 antigen contributes to the immuneresponse (see below), strongly indicating that the antigen is presentand actively involved in the immune response.

Example 4 Immunogenicity of Vectors Encoding M tuberculosis Antigens inMice

First, the immunogenicity of the adapter plasmids as described inexample 1 (DNA constructs) was studied in mice. The constructs encodedone, two or three TB antigens: Ag85A, Ag85B and TB10.4. The DNAconstructs encoding for the multiple TB antigens were designed in twoways as described above, i.e. expressing a polyprotein comprising directfusions not containing the myc tag and expressing a polyproteincomprising a sequence encoding a protease and the protease recognitionsites resulting in the cleavage of the polyprotein (also not containingthe myc tag) into discrete polypeptides. The following DNA constructswere used (see example 1):

Single antigen constructs TB-5 (Ag85A), TB-6 (Ag85B) and TB-7 (TB1O.4)Double antigen constructs

TB-3 (ALV-dig*-Ag85A-dig-Ag85B) and TB-4 (Ag85A-Ag85B direct 25 fusion)

Triple antigen constructs

TB-L (ALV-dig*-Ag85A-dig-g85B-dig-TB1O4) and TB-S (Ag85A-Ag85B-TB1O.4direct fusion).

The experimental set up is given in FIG. 11. Seven groups of mice wereimmunized with individual TB DNA constructs (two experiements, seebelow). For each immunization, DNA was injected intramuscularly threetimes (3×50 μg) with intervals of 2.5 weeks. As a negative control, onegroup of mice received three injections of PBS. Additional control groupreceived single dose of 6×10⁵ cfu BCG (strain SSI1331) subcutanously.

One week after the last DNA immunization, and six weeks after the BCGimmunization the mice were sacrificed. Spleens were isolated to serve asa source of cells for cellular immunological assays. Sera, required forhumoral response analysis, were collected by heart punction and pooledper group.

The level of specific cellular immune response was determined usingintracellular IFNγ staining (ICS) FACS assay, by measuring the frequencyof IFNγ+ CD4+ and IFNγ+ CD8+ splenocytes after in vitro re-stimulationwith peptide pools of corresponding antigens. The immune sera weretested using immunofluorescence of A549 cells transduced with adenovirusencoding for corresponding antigen.

Two independent immunization experiments were performed. For the firstexperiment, 3 mice per group were used and the immune response wasanalyzed for each mouse individually. For the second experiment, 8 miceper group were used for DNA immunizations and 4 mice per group forcontrol immunizations. After in vitro stimulation with peptides, samplesof two-by-two mice from the same group were pooled and stained for FACSanalysis. Similar results were obtained in both experiments and the datawere brought together for statistical analysis.

The intracellular IFNγ staining (ICS) was performed as follows.Splenocytes (10⁶ per well of 96-well plate) were stimulated in duplicatewith appropriate peptide pool as indicated (final concentration 2 pg/mlper peptide), in the presence of co-stimulatory antibodies:anti-mouse-CD49d and anti-mouse-CD28 (Pharmingen) in a final dilution of1:1000. Peptide pools consisted of 15-mer peptides spanning wholeantigens, with 10-mer (Ag84B) or 1′-mer (Ag85A, TB10.4) overlappingsequences, or adjusted for Ag85B with peptide p1 and p2 from Ag85A, asoutlined below in example 6 and 7. Samples from BCG and PBS immunizedmice were stimulated additionally with CFP (Culture Filtrate Protein;final concentration 10 pg/ml) and PPD (Purified Protein Derivative;final concentration 10 pg/ml), which are antigens commonly used for invitro stimulation upon BCG immunization. As a positive control, sampleswere stimulated with PMA/ionomycin (final concentrations: 50 ng/ml and 2μg/ml, respectively) whereas the incubation with medium served as anegative control (no stimulation). After 1 h stimulation at 37° C.,secretion blocker GolgiPlug was added (Pharmingen; final dilution 1:200)and the incubation was continued for an additional time period of 5 h.The corresponding duplicate samples were pooled and processed for FACSanalysis. Briefly, cells were washed with PBS containing 0.5% BSA andincubated with FcR Blocker (Pharmingen; dilution 1:50) for 10 min onice. After a washing step, the cells were incubated with CD4-FITC(Pharmingen; dilution 1:250) and CD8-APC (Pharmingen; dilution 1:50) for30 min on ice. Upon washing cells were fixed and permabilized withCytofix/Cytoperm (Pharmingen) for 20 min on ice, followed by a washingstep with Perm/Wash buffer (Pharmingen). Intracellular IFNγ was stainedusing anti-IFNγ-PE (Pharmingen; dilution 1:100) for 30 min on ice. Afterfinal washing steps cells were resuspended in CellFix (BD) and analyzedusing flow cytometer. At least 10,000 CD8+ cells were measured for eachindividual sample. Results are expressed as a percentage of CD4+ or CD8+cells that express IFNγ.

An overview of the in vitro re-stimulation samples is given in Table II.The results of the ICS are presented in FIG. 12-16.

TABLE II Overview of the in vitro re-stimulation samples. In vitroantigen stimulation Immunization Ad85A Ad85B TB10.4 CFP PPD PMA MediumAd85A (TB-5) X X X X Ad85B (TB-6) X X X X TB10.4 (TB-7) X X XAd85A.Ad85B (TB-3) X X X X Ad85A.Ad85B (TB-4) X X X X Ad85A.Ad85B.TB10.4(TB-L) X X X X X Ad85A.Ad85B.TB10.4 (TB-S) X X X X X BCG X X X X X X XPBS X X X X X X X

FIGS. 12A and B show that background levels were very low when the cellswere not stimulated. FIG. 13A shows a high frequency of IFNγ+ CD4+splenocytes after stimulation with peptides of the Ag85A pool. There isa clear cross-reactivity with CD4+ cells obtained from mice injectedwith the construct harboring the Ag85B encoding gene, which is notunexpected due to the high structural homology between Ag85A and Ag85B.In contrast to what was found for CD4+ cells, no stimulation of CD8+splenocytes (see FIG. 13B) was detected of cells from mice injected withconstructs encoding either Ag85A alone or in the context of Ag85B (lanesAg85A, Ag85B, TB-3L and TB-4S). However, there was a striking increasein IFNγ+ CD8+ splenocytes in mice injected with the triple constructsTB-L and TB-S, clearly indicating an important role of the additionalantigen (TB10.4) present in these constructs. Apparently, in thissetting, the TB10.4 antigen is able to strongly increase the frequencyof CD8+ splenocytes reactive towards the Ag85A peptides, where Ag85Aalone (or in combination with Ag85B) provides no responses. FIG. 14Ashows that Ag85B in all settings in which it was present is able toincrease the frequency of IFNγ+ CD4+ splenocytes, whereas the effect onIFNγ+ CD8+ splenocytes is minimal (see FIG. 14B). Also here,cross-reactivity is found between Ag85B and Ag85A (FIG. 14A) asdiscussed above. FIG. 15A shows that the frequency of IFNγ+ CD4+splenocytes responding to the TB10.4 related peptide pool is present,where no real difference can be found between mice injected with eithera construct with TB10.4 alone or a construct comprising the tripleinserts. However, as shown in FIG. 15B, the frequency of IFNγ+ CD8+splenocytes from mice that were injected with constructs comprising thegene encoding the TB10.4 antigen, is dramatically increased uponstimulation with TB10.4 related peptides, especially in the context ofthe triple inserts (Note the y-axis, indicating that an average of 1.5%of the splenocytes was reactive). The results are summarized in FIG. 16A(triple insert in TB-L: with protease and protease digestion sites) andFIG. 16B (TB-S: direct linked antigens). Clearly, the different antigenscontribute in different manners to the immune response: Ag85A inducesboth CD4 and CD8 responses; Ag85B only induces a strong CD4 response andhardly any CD8 response. In contrast to Ag85B, the TB10.4 antigeninvokes a strong CD8 response and a minor CD4 response. This indicatesthe clear beneficial subsidiary effect of the different antigens encodedby the sequences present in the triple inserts.

The BCG immunization did not result in significant ICS response.However, splenocytes of BCG immunized mice did produce high levels ofIFNγ after 72 h stimulation with CFP or PPD, as determine using an IFNγELISA kit, which indicates that mice were immunized efficiently (datanot shown).

To determine whether any antigen-specific antibodies were actuallyraised in the mice injected with the different DNA constructs, A549cells were transduced with Ad35 recombinant adenoviruses encoding the TBantigens in 96 well plates. The adenoviruses were produced as describedin example 2. For this, 1×10⁴ cells were seeded per well and viruseswere infected with a multiplicity of infection of 5000. Two days afterinfection cells were fixed with Cytofix/Cytoperm (20 min at 4° C.),followed by a washing step with Perm/Wash buffer. Cells were incubatedwith immunized mice sera, diluted 1:2 in Perm/Wash buffer, for 1 h at37° C. Upon washing, goat anti-mouse-FITC, diluted 1:5 in Perm/Washbuffer, was added and incubated for 30 min at 37° C. After a final wash,cells were analyzed using a fluorescence microscope.

The immunofluorescence analysis revealed strong antigen specificstaining of cells with sera obtained from mice immunized with TB-6(Ag85B alone), TB-3 (ALV-dig*-Ag85A-dig-Ag85B), TB-4 (Ag85A-Ag85B directfusion) and TB-L (ALV-dig*-Ag85A-dig-Ag85B-dig-TB10.4). Weak stainingwas observed with sera from mice immunized with TB-S (Ag85A-Ag85B-TB10.4direct fusion), while sera obtained upon immunization with TB-5 (Ag85Aalone) and TB-7 (TB10.4 alone) did not exhibit any staining. Thisindicates that at least some of the antigens are able to elicit anantibody response. Full cleavage of the protease from the remaining partof the polyprotein and expression levels of the separate antigens wasnot determined in this experiment.

Example 5 Construction of rAd Vectors Encoding an Antigen and anAdjuvant

Here, a novel recombinant replication-defective adenoviral vector isconstructed, herein designated Ad35-X-A1_(K63), which co-expresses anantigen (referred to as X) and a mutant derivative of CtxA1 that harborsa lysine substitution at amino acid no. 63 (i.e. herein referred to asA1_(K63)) in place of the serine that is present in the parental CtxA1.

The construction of adapter plasmids suitable to generate E1-deletedAd35-based vectors capable of expressing X and A1_(K63) is achieved byintroducing PCR-amplified X using standard PCR procedures known topersons skilled in the art, and introducing appropriate cloningrestriction sites. The resultant PCR-generated DNA fragment is digestedwith the respective restriction endonuclease(s) and annealed to anadapter plasmid generally as described above for Ag85A, Ag85 and TB10.4.Additional analysis by restriction endonuclease digestion, PCR andsequencing of the cloned PCR fragment are conducted to verify that theDNA was not altered during construction.

DNA encoding A1_(K63) is amplified from plasmid pOGL1-A comprising acopy of CtxA1. The nucleotide sequence of ctxA1 is available in GenBank(Accession # A16422) and modified by replacing the serine-63 TCA codon(nt 187-189) with a lysine codon AAA. The mutant derivative is generatedusing the QuikChange® Site-Directed Mutagenesis Kit (Stratagene). Thesite-directed mutagenesis process entails whole-plasmid PCR usingpOGL1-A1 DNA as template, forward primer 5′-TGT TTC CCA CCA AAA TTA GTTTGA GAA GTG C-3′ (SEQ ID NO:24) and reverse primer 5′-CAA ACT AAT TTTGGT GGA AAC ATA TCC ATC-3′ (SEQ ID NO:25). This procedure modifiesnucleotides 187-189 by replacing the TCA codon with an AAA codon (seeunderlined sequences). The resultant PCR-generated plasmid is digestedwith DpnI to remove the template DNA and the digested DNA is introducedinto E. coli by chemical transformation and grown on agar at 30° C. for16 hr. Isolated colonies are selected and DNA was extracted fromovernight liquid cultures. Plasmid PCR using primers specific forA1_(K63), and agarose gel electrophoresis are conducted to screen for anappropriate derivative. The mutant insert is cloned thereafter into thesame adapter plasmid that contains X, either upstream or downstream of Xand adenoviruses are produced as described above.

Example 6 Dose Response of Ad35.TB Vectors in Mice

Ad35 recombinant viruses expressing the different single and fused TBantigens were used to test antigenicity in mice. Methods to quantifyT-cells that produce interferon-gamma (IFN-γ) after stimulation withproteins or peptide pools are typically performed using methods known inthe art and for example described in WO 2004/037294, Sander et al.(1991) and Jung et al. (1993). Details are described below.

The triple insert vector TB-S was used in a dose-response immunogenicitytest. Four different doses of viral particles (10⁷, 10⁸, 10⁹ and 10¹⁰vp) were injected intramuscularly in different groups of C57BL/6 mice (5mice per group), whereas 3 mice served as a negative control and wereinjected with 10¹⁰ vp of the empty viral vector. Two weeks afterimmunization, the mice were sacrificed and splenocytes were isolated toserve as the source of cells for cellular immunological studies. Thelevel of antigen-specific cellular immune responses was determined usingthe intracellular IFNγ staining (ICS) FACS assay, by measuring thefrequency of IFNγ CD4+ and CD8+ splenocytes as described above. Theresults are shown in FIG. 17. Clearly, the Ad35 based TB-S vectorinduces an antigen specific immune response in a dose dependent manner,especially in relation to the increase in response with Ag85B specificCD4 cells (FIG. 17C). No response was found related to TB10.4 specificCD4 cells (FIG. 17E), and no response was found related to Ag85Bspecific CD8 cells (FIG. 17D). While hardly any responses were detectedwith the 10⁷ and 10⁸ doses in respect to Ag85A and TB10.4 specific CD8cells (FIGS. 17B and 17F respectively), a marked increase was foundusing the 10⁹ and 10¹⁰ doses. The 1 dose did not give any significanteffects in any of the settings, while the 10⁸ dose also resulted in anincrease in Ag85A and Ag85B specific CD4 cells (FIGS. 17A and 17Crespectively). Similar data were found using the TB-L construct (datanot shown).

During an assessment for the CD8 immunodominant sequence epitope mappingof M. tuberculosis antigens in mice, it was found that the peptidesreferred to as p1 (FSRPGLPVEYLQVPS; SEQ ID NO:26) and p2(GLPVEYLQVPSPSMG; SEQ ID NO:27) of Ag85A were the only CD8immunodominant epitopes for C57BL/6 mice. The underlined stretch shouldtheoretically fit in the MHC molecules of C57BL/6 mice. The sequence ofthe Ag85A antigen in this region of the protein (amino acids 1-19:FSRPGLPVEYLQVPSPSMG; SEQ ID NO:28) is identical to the sequence of Ag85Bin the same region. However, the peptides p1 and p2 from the Ag85B pool,although comprised of the same sequence as peptides from Ag85A, did notgive any CD8 response (see FIG. 17D). This suggests that the peptides p1and p2 from Ag85B were not in order, perhaps due to production effectsor contaminations. Therefore, an additional dose response experiment wasperformed in which the in vitro stimulation peptide pool of Ag85B wasreconstituted with p1 and p2 from the Ag85A pool. The experiment wasperformed with both TB-S and TB-L vectors, using doses of 10⁷, 10⁸, 10⁹and 10¹⁰ vp. The T cell response was determined two weeks afterimmunization, generally as described above. As negative controls, onegroup of mice was injected with PBS, while one group was injected withan empty Ad35 virus (10¹⁰ vp). The results with respect to CD8 cells arepresented in FIG. 17G (TB-L, left graph, TB-S, right graph). Clearly,CD8 positive cells were measured upon in vitro stimulation with theadjusted Ag85B pool, although the peptides from the Ag85A antigen wereidentical to the peptides of the Ag85B antigen, which were originallyused and did not provide any positive results. These observationsnevertheless show that also the Ag85B protein as encoded by theAd35-based adenoviruses can induce a CD8 positive T cell response afterinfection of said viruses.

Example 7 Ad35 Based TB Vectors Used as a Boost Upon Priming with BCG

In another experiment Ad35 vectors expressing TB antigens were tested asa boosting agent for BCG immunization. Hereto, groups of mice wereinjected subcutaneous with BCG vaccine (Bacilli Calmette-Guerin;reference standard FDA and generally known in the art of tuberculosisvaccination) according to protocols delivered by the FDA (standards andtesting section CBER).

Four groups of mice (8 mice per group) were primed with BCG (6×10⁵cfu/mouse) subcutaneously ten weeks prior to infection with theadenoviral vectors based on Ad35 carrying the three directly linked TBantigens (TB-S) or with the adenoviral Ad35 vectors carrying thefollowing combinations of antigens:

-   -   TB-4 alone (comprising the Ag85A and Ag85B direct fusion)    -   TB-4+TB-7 (comprising TB10.4 alone)    -   TB-5 (comprising Ag85A alone)+TB-6 (comprising Ag85B        alone)+TB-7.        Two control groups (4 mice per group) were primed with PBS or        with BCG, whereafter the PBS group received PBS as        mock-immunization, and the BCG primed control group received 10⁹        vp of the empty Ad35 vector. Injections with the Ad35 based        vectors were performed in all cases with 10⁹ vp,        intramuscularly. Four weeks post-infection (14 weeks after        prime), mice were sacrificed and splenocytes were isolated and        used as described above. The results are shown in FIG. 18. The        presence of the Ag85A antigen resulted in a significant effect        towards Ag85A specific CD4 cells (FIG. 18A). As expected (see        also FIG. 13B), the triple construct TB-S induced an Ag85A        specific CD8 response, while the TB-4 vector did not induce such        a response (FIG. 18B). Similar results were found earlier (FIG.        13B), indicating that the presence of Ag85A alone or in        combination with Ag85B does not give a CD8 response, whereas        such a response is found when TB10.4 is present. Interestingly,        no effect was determined when the separate vectors were injected        but in a single shot (TB-4/TB-7 or TB-5/TB-6/TB-7 in FIG. 18B),        indicating that the TB10.4 antigen can not induce an Ag85A        specific CD8 response when co-injected, but rather that the        antigen should be present in the same construct or at least in        the same cell. The mechanism for the adjuvant effect of TB10.4        is yet unclear.

The effects seen with the Ag85B antigen are in concert with what wasfound earlier (FIGS. 18C and D). It must be noted that the presence ofthe TB10.4 antigen in the triple construct TB-S does not give rise to aAg85B-specific CD8 response, in contrast to what is found with Ag85A.Both antigens are well expressed from the constructs, as was shown inFIG. 10B. The negative effect may be due to a corrupted peptide poolused to measure any CD8 response towards Ag85B (see example 6 andbelow).

The induction of CD4+ cells using TB10.4 is very low (FIG. 18E). Theinduction of CD8+ cells using TB10.4 in a separate vector(TB-5/TB-6/TB-7) is significant (note the scale on the y-axis; see alsoFIG. 15B). The induction of TB10.4 specific CD8 cells using TB-S is veryhigh (FIG. 18F), with an average of around 12% IFNγ positive CD8 cells.

It can be concluded that the TB10.4 antigen is capable of inducing a CD8response towards an antigen, which as a single construct does not giverise to a CD8 response (Ag85A). It is known that activation of CD8 cellsrequires a somewhat higher antigenic threshold than the activation ofCD4 cells, which is at least partly due to complex machinery involved inantigen processing and presentation by MHC class 1 molecules (Storin andBachmann. 2004). Here, it was found that when TB10.4 was coupled toantigens Ag85A and Ag85B in a triple-antigen construct, strong CD8responses were triggered, not only against TB10.4 itself but alsoagainst Ag85A. It is likely that the physical presence of TB10.4 in theconstruct increases the efficiency of transport of the fusion protein tothe proteosome, which is necessary for the efficient presentation to andactivation of CD8 cells. The reason for the higher TB10.4-specific CD8cell response is most likely due to an increased expression level of thetriple construct in comparison to the vector carrying the TB10.4 antigenalone. Although the CD8 response towards TB10.4 alone was alsosignificant, no expression levels of TB10.4 could be determined due tolack of TB10.4 specific antisera for western blotting.

The increased targeting to the proteosome might be the result of thepresence of specific sites in TB10.4 molecule, such as sequencesinvolved in binding of ubiquitin (or other molecules responsible forlabeling the proteins destined for processing), or transporter proteins,or sequences that otherwise increase processing and presentation in thecontext of MHC class I molecules (Wang et al. 2004). Alternatively, thepresence of TB10.4 protein in the construct might physically destabilizethe fusion protein, leading to increased degradation rate of themolecule. Increased level of antigen processing leads in general toincreased CD8 cell activation. Furthermore, if much protein ends up inthe proteosome for class I presentation, less will be present in cytosoland extracellularly and, thus, not be available for activation of Bcells. It has been reported that an inverse correlation exists betweenantigen processing (i.e. CD8 activation) and antigen specific antibodytiter (Delogu et al. 2000). It is interesting to mention that a muchstronger antigen-immunoflurescence was observed in sera from miceimmunized with double-antigen constructs rather than from thetriple-antigen construct immunized mice. This finding suggests that ourtriple-antigen molecules, containing TB10.4, are highly susceptible toprotesome degradation and CD8 cell activation and, thus, less availablefor antibody induction. As a strong T cell response is a preferableresponse against tuberculosis, it is concluded that an Ad35-basedtriple-antigen vector, which comprises a nucleic acid encoding theTB10.4 antigen and at least one other TB antigen, preferably Ag85A andmore preferably, both Ag85A and Ag85B, is very suited to be used in avaccine against tuberculosis. The found effects may be even furtherincreased by using BCG as a priming agent, as indicated by the resultsshown in FIG. 18.

Using the new peptide pool for Ag85B with the peptides p1 and p2 ofAg85A added (as described in example 6), also the prime/boost study withBCG prime, Ad35-TB boost was repeated, although now the splenocytes wereremoved from mice that were sacrificed 26 weeks after prime (16 weeksafter immunization). Mice (8 per group) were immunized with PBS,Ad35.Empty, Ad35.TB-S, or Ad35.TB-L with either 10⁹ or 10¹⁰ vp of therespective viral vectors. Results are shown in FIG. 25 (Ag85Astimulation), 26 (Ag85B stimulation) and 27 (TB10.4 stimulation). Theresults clearly indicate that significant CD4 and CD8 responses canstill be measured after prolonged period of time.

Example 8 Prime-Boost-Challenge Experiment in Guinea Pigs

In a subsequent experiment, it was investigated whether priming withBCG, followed by a boost with Ad35-based TB vectors, would protectagainst a Mycobacterium tuberculosis infection in a challenging set-up.

Guinea pigs were initially primed with BCG typically as indicated above(6×10⁵ cfu per animal). After 14 weeks, the animals were eitherimmunized with 10¹⁰ vp Ad35.TB-S (Ag85A-Ag85B-TB10.4) or Ad35.TB-4(Ag85A-Ag85B) recombinant viruses, or injected with PBS (control group).Eight weeks later, the animals were challenged with ˜100 cfu M.tuberculosis per animal. The animals are monitored up to approximately78 weeks post prime for survival. Intermediate observations suggest thatthe BCG prime followed by an Ad35-TB boost ensures a higher survivalrate than BCG alone.

REFERENCES

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1. A recombinant replication-defective adenovirus comprising a nucleicacid sequence encoding two or more antigens from at least onetuberculosis (TB)-causing bacillus.
 2. The recombinantreplication-defective adenovirus according to claim 1, wherein saidrecombinant replication-defective adenovirus is selected from the groupconsisting of human adenovirus serotypes Ad11, Ad24, Ad26, Ad34, Ad35,Ad48, Ad49 and Ad50.
 3. The recombinant replication-defective adenovirusaccording to claim 1, wherein said TB-causing bacillus is Mycobacteriumtuberculosis, Mycobacterium africanum and/or Mycobacterium bovis.
 4. Therecombinant replication-defective adenovirus according to claim 1,wherein said nucleic acid sequence encodes at least two antigensselected from the group consisting of antigens encoded by the Ag85A,Ag85B, and TB10.4 open reading frames of M. tuberculosis.
 5. Therecombinant replication-defective adenovirus according to claim 1,wherein at least two of said antigens are expressed from onepolyprotein.
 6. The recombinant replication-defective adenovirus ofclaim 1, wherein at least two of said antigens are linked so as to forma fusion protein.
 7. The recombinant replication-defective adenovirusaccording to claim 4, wherein said nucleic acid sequence encodes allthree antigens Ag85A, Ag85B and TB10.4, cloned in that 5′ to 3′ order.8. A recombinant polynucleotide vector comprising a nucleic acidsequence encoding two or more antigens and a protease-recognition site,wherein said antigens are expressed as a polyprotein, said polyproteincomprising the protease-recognition site separating at least two of thetwo or more antigens.
 9. The recombinant polynucleotide vector accordingto claim 8, wherein said polynucleotide vector is packaged into areplication-defective adenovirus and wherein said replication-defectiveadenovirus is selected from the group consisting of human adenovirusserotypes Ad11, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50.
 10. Therecombinant polynucleotide vector according to claim 8, wherein saidnucleic acid sequence further comprises a sequence encoding a protease.11. The recombinant polynucleotide vector according to claim 10, whereinsaid protease, upon expression, is expressed as part of the polyproteinand is linked to at least one of said antigens by a protease-recognitionsite.
 12. The recombinant polynucleotide vector according to claim 11,wherein said protease-recognition site comprises a sequence according toSEQ ID NO:21 or
 22. 13. The recombinant polynucleotide vector accordingto claim 10, wherein said protease is from an Avian Leukosis Virus(ALV).
 14. The recombinant polynucleotide vector according to claim 8,wherein said antigens are from at least one tuberculosis (TB)-causingbacillus.
 15. The recombinant polynucleotide vector according to claim14, wherein said TB-causing bacillus is Mycobacterium tuberculosis,Mycobacterium africanum and/or Mycobacterium bovis.
 16. The recombinantpolynucleotide vector according to claim 8, wherein said nucleic acidsequence encodes at least two antigens encoded by the Ag85A, Ag85B, andTB10.4 open reading frames of M. tuberculosis.
 17. The recombinantpolynucleotide vector according to claim 16, wherein said nucleic acidsequence encodes the antigens Ag85A, Ag85B and TB10.4, and wherein thenucleic acid sequences encoding said antigens are cloned in that 5′ to3′ order.
 18. A recombinant polynucleotide vector comprising a nucleicacid sequence encoding an antigen and a genetic adjuvant, wherein saidantigen and said genetic adjuvant are linked.
 19. The recombinantpolynucleotide vector according to claim 18, wherein said polynucleotidevector is packaged into a replication-defective adenovirus.
 20. Therecombinant polynucleotide vector according to claim 19, wherein saidadenovirus is selected from the group consisting of human adenovirusserotypes Ad11, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50.
 21. Therecombinant polynucleotide vector according to claim 18, wherein saidnucleic acid sequence further comprises a sequence encoding a protease.22. The recombinant polynucleotide vector according to claim 21, whereinsaid protease is linked to said antigen and/or to said genetic adjuvantby a second protease-recognition site.
 23. The recombinantpolynucleotide vector according to claim 22, wherein said secondprotease-recognition site comprises a sequence according to SEQ IDNO:22.
 24. The recombinant polynucleotide vector according to claim 19,wherein said first protease-recognition site comprises a sequenceaccording to SEQ ID NO:21.
 25. The recombinant polynucleotide vectoraccording to claim 21, wherein said protease is from an Avian LeukosisVirus (ALV).
 26. The recombinant polynucleotide vector according toclaim 18, wherein said antigen is from a tuberculosis (TB)-causingbacillus.
 27. The recombinant polynucleotide vector according to claim26, wherein said TB-causing bacillus is Mycobacterium tuberculosis,Mycobacterium africanum or Mycobacterium bovis.
 28. The recombinantpolynucleotide vector according to claim 18, wherein said antigen isselected from the group consisting of antigens encoded by Ag85A, Ag85B,and TB10.4 open reading frames of M. tuberculosis.
 29. The recombinantpolynucleotide vector according to claim 18, wherein said geneticadjuvant comprises a cholera toxin (CtxA1) or a mutant derivativethereof, said mutant derivative comprising a serine to lysinesubstitution at amino acid position 63 (A1_(K63)).
 30. A multivalent TBvaccine comprising: the recombinant adenovirus claim 1, and apharmaceutically acceptable excipient.
 31. An improvement in a geneticadjuvant, the improvement comprising using Mycobacterium antigen TB10.4as the genetic adjuvant.
 32. A composition comprising: Mycobacteriumantigen TB10.4 for the treatment or prophylaxis of a disease in whichthe immune response of a host towards a certain antigen or therapeuticcomponent of interest needs to be stimulated.